Methods for detecting acetohydroxyacid synthase inhibitors

ABSTRACT

The invention provides a method for determining whether a compound inhibits acetohydroxyacid synthase. The invention further provides a method for determining whether a plant is resistant to an acetohydroxyacid synthase inhibiting compound.

This is an application for Reissue of U.S. Pat. No. 5,356,789, issuedOct. 18, 1994 on U.S. application Ser. No. 08/068,458 filed May 28,1993.

BACKGROUND OF THE INVENTION

The pathway leading to the biosynthesis of branched chain amino acids(valine, leucine and isoleucine) is vital to the survival of plants.This pathway, and the enzymes promoting it, is vulnerable to severalclasses of highly potent herbicides including imadazolinones,sulfonylureas, sulfonamides and pyrimidyloxybenzoates. Such herbicidesact by inhibiting acetohydroxyacid synthase (AHAS), the first enzymefunctioning in the pathway.

Recently, weed populations have been discovered which are resistant toAHAS-inhibiting herbicides (D. L. Shaner, Herbicide Resistance in Weedsand Crops, ed. J. C. Caseley, G. W. Cussans and R. K. Atkin (Oxford:Butterworth-Heinemann, 1991), 187-198). These resistant biotypes containan altered AHAS enzyme which is no longer inhibited by these herbicides.Once such a resistant weed population has developed, the weed managementprogram has to be changed to prevent the propagation of the resistantweed. Therefore, a method to rapidly determine if a weed population hasdeveloped resistance to an AHAS-inhibiting herbicide would have greatutility and would permit the agriculturalist to adapt his weedmanagement program to control the resistant weed more effectively.

Because several classes of AHAS inhibiting compounds are highly potentherbicides, there is an ongoing search to discover new and moreeffective AHAS inhibitors. To identify these new inhibitors, assays areused to measure the extent of AHAS inhibition caused by the compounds'use. However, the assays currently employed are often arduous, expensiveand/or time-consuming.

It is an object of the present invention to provide a method fordetermining whether a compound inhibits acetohydroxyacid synthase.

It is also an object of the present invention to provide a method fordetermining whether a plant is resistant to an acetohydroxyacid synthaseinhibitor,

These and other objects of the present invention will become moreapparent from the detailed description thereof set forth below.

SUMMARY OF THE INVENTION

The present invention relates to a method for determining whether acompound inhibits acetohydroxyacid synthase (AHAS). In particular, thepresent invention relates to a method for determining whether a compoundinhibits the AHAS enzyme by detecting the presence of and measuring thequantity of its condensation products in a biological system.

The present invention also relates to a novel in vivo method fordetermining whether a plant is resistant to an AHAS inhibitor. Inparticular, the present invention relates to a method for determiningwhether a plant is AHAS-inhibitor resistant by detecting the presence ofand measuring the quantity of its condensation products in thebiological system and comparing the results to a predetermined standard.

DETAILED DESCRIPTION OF THE INVENTION

Weeds cause tremendous global economic losses by reducing crop yieldsand lowering crop quality. Several classes of highly potent herbicideseffectively control weeds by inhibiting AHAS. Therefore, there is anongoing search to discover new and more effective AHAS inhibitors. Toidentify these new inhibitors, assays can be used to measure theproducts of AHAS activity. However, the assays currently employed areoften arduous, expensive and/or time-consuming.

New herbicides have been discovered which inhibit ketol-acidreductoisomerase (KARI), the enzyme which immediately follows AHAS inthe branched chain amino acid pathway (J. V. Schloss and A. Aulabaugh,Biosynthesis of Branched Chain Amino Acids, ed. Z. Barak, D. M. Chipmanand J. V. Schloss (New York: VCH Publishers, 1990), 329-356). Plantstreated with these herbicides accumulate acetolactate andacetohydroxybutyrate (AL/AHB), the condensation products of AHAS. Theamount of AL/AHB present in the plants can then by measured to determinethe extent of inhibition.

Advantageously, the present invention provides a rapid and quantitativemethod for determining whether a compound inhibits AHAS. The methodcomprises treating an AHAS inhibitor-susceptible plant or plant partwith a biologically active compound and an effective amount of a KARIinhibitor; treating the AHAS inhibitor-susceptible plant or plant partwith the KARI inhibitor alone; and measuring the amount of AL and AHBpresent in the treated plants or plant parts to determine if the amountof AL and AHB present in the plant or plant part treated with thebiologically active compound and the KARI inhibitor is less than theamount of AL and AHB present in the plant or plant part treated with theKARI inhibitor alone. Such a result is characteristic of an AHASinhibiting compound.

Advantageously, the present invention also provides a rapid andquantitative method for determining whether a plant is resistant to anAHAS inhibitor. This method comprises treating a plant or a part of theplant with an effective amount of an AHAS inhibitor and an effectiveamount of a KARI inhibitor; treating the plant or plant part with aneffective amount of the KARI inhibitor alone; and measuring the amountof AL and AHB present in the treated plants or plant parts to determineif the amount of AL and AHB present in the plant or plant part treatedwith the AHAS inhibitor and the KARI inhibitor correlates with apredetermined AHAS-inhibitor resistance standard. For example, where aneffective amount of an AHAS inhibitor and an effective amount of a KARIinhibitor produce a result of at least about 15% of the amount of AL andAHB present in the plant or plant part treated with the KARI inhibitoralone, the plant is essentially AHAS inhibitor resistant. It has beendiscovered that heterozygous resistant plants treated with an AHASinhibitor and a KARI inhibitor have about 15% to 50% of the amount of ALand AHB present in the KARI inhibitor-treated plants, and homozygousresistant plants treated with an AHAS inhibitor and a KARI inhibitorhave more than 50% of the amount of AL and AHB present in the KARIinhibitor treated plants.

Advantageously, the present invention may be used to determine if weedsare resistant to AHAS inhibitors. Because of the rapidity of theinvention methods, critical decisions can be made on how to treatuncontrolled weeds after AHAS inhibitor applications.

Beneficially, the methods of the present invention can be used in thefield or laboratory with minimal equipment. And the methods of thisinvention are more economical and significantly less time-consuming thanthe enzyme extraction and green house spray procedures currentlyemployed.

KARI inhibitors which are suitable for use in the methods of the presentinvention include (dimethylphosphinyl)glycolic acid;2-(dimethylphosphinoyl)-2-hydroxyacetic acid; sodiumN-hydroxy-N-alkyloxamates and sodium N-hydroxy-N-aralkyloxamates. Forthe N-hydroxy-N-alkyloxamates, the alkyl group is preferably C₁ -C₆alkyl or C₃ -C₇ cycloalkyl, most preferably isopropyl. A preferredaralkyl group is benzyl.

AHAS inhibitors which are suitable for use in the method used to detectresistance include

a) imidazolinone inhibitors such as

5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid(imazethapyr);

2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylicacid (imazaquin);

isopropylammonium2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate (imazapyr);

methyl 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate; and

2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid;

b) sulfonylurea inhibitors such as

1-(2-chlorophenylsulfonyl)-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea(chlorsulfuron);

methyl 2- 3-(4,6-dimethylpyrimidin-2-yl)ureidosulfonyl!benzoate(sulfometuron-methyl); and

methyl o-{{3-4,6-bis(difluoromethoxy)-2-pyrimidinyl!ureido}sulfonyl}benzoate;

c) sulfonamide inhibitors such as

N-(2,6-difluorophenyl)-5-methyl-1,2,4-triazolo1,5-a!pyrimidine-2-sulfonamide (flumetsulam); and

N-(2,6-dichlorophenyl)-5-methyl-1,2,4-triazolo1,5-a!pyrimidine-2-sulfonamide; and

d) pyrimidyloxybenzoate inhibitors such as

o- (4,6-dimethoxy-2-pyrimidinyl)oxy!benzoic acid.

Plant species suitable for use in the methods of the present inventioninclude monocotyledonous plant species such as Johnsongrass, blackgrassand corn, and dicotyledonous plant species such as pigweed, morningglory, chickweed, sunflower, tobacco and lima bean. Plant parts suitablefor use in the methods of the invention include young rapidly growingtissue, such as meristematic tissue, with young rapidly growing shoots,leaves, roots and flower buds being preferred.

In the methods of the present invention, the plants or plant parts arepreferably treated with an effective amount of an inhibitor or testcompound. One of ordinary skill in the art would be able to determinethe effective amount of any particular test compound by routineexperimentation. For example, about a 1 μM to 1,000 μM, more preferablyabout 5 μM to 500 μM, most preferably about 10 μM to 100 μM solution ofthe test compound should be employed, depending on the potency. Thesolution containing the test compound may be combined with a 1 μM to1,000 μM solution, preferably about 5 μM to 500 μM, most preferablyabout 10 μM to 100 μM, of the KARI inhibitor; and/or a 1 μM to 1,000 μMsolution, preferably about 5 μM to 500 μM, most preferably about 10 μMto 100 μM, of the AHAS inhibitor. Of course higher concentrations wouldbe similarly effective but may be wasteful and are usually notnecessary.

The amount of AL and AHB present in the treated plants and plant partsmay be determined according to known procedures in the art such as themethod of Westerfeld (J. Biol. Chem, 161, 495-502 (1945)) or gas liquidchromatography.

To determine the amount of AL and AHB present in a plant or plant partaccording to the method of Westerfeld, the AL and AHB present in theplant or plant part is extracted into water; the water extract istreated with sulfuric acid; the acidified water extract solution istreated with a 0.5% creatine solution and a 5% a-naphthol in sodiumhydroxide solution; and the color of the resulting solution is measuredto determine the amount of AL and AHB present in the treated plant orplant part.

In order to facilitate a further understanding of the invention, thefollowing examples are presented to illustrate more specific detailsthereof. The invention is not to be limited thereby as the full scope ofthe invention is defined in the claims.

EXAMPLE 1 Evaluation of In Vivo Inhibition of Acetohydroxyacid SynthasePlant Material

Corn (Zea mays) (Pioneer var. 3475) and sunflower (Helianthus annuus)(Dahlgran var. DO-164) seeds are germinated and grown for 6 days incylinders of germination paper that are kept moist by standing thecylinders in distilled water in a 1 L beaker covered with a perforatedplastic bag. Seedlings are transferred to 120 mL plastic containers andgrown hydroponically in a modified nutrient solution. All plants aregrown in a growth chamber (13 h photoperiod; 28°/22° C.; 300 μE/m²/sec). Corn is treated when the fourth leaf is just emerging from thewhorl and sunflowers when the third leaf is emerging.Sulfonylurea-resistant and sensitive chickweed (Stellaria media) aregrown from seed in the greenhouse until the plants reach the 2 to 4 leafstage.

Treatments

Stock solutions of 10 μM technical (dimethylphosphinyl)glycolic acid aremade by dissolving 1.52 mg/mL in water and are kept frozen until use.Solutions of different concentrations are made by dilution from thisstock solution. Ten μM stock solutions of imazaquin andsulfometuron-methyl are made by dissolving 3.11 and 3.64 mg/mL oftechnical material, respectively, in a suitable organic solvent, such asacetone or tetrahydrofuran, and dilutions made from these stocksolutions. Technical grade amino acids are dissolved directly inapplication solutions to the desired final concentrations.

In excised shoot (plant part) experiments, shoots are cut free from theroots under degassed water and the excised shoots are placed in vialscontaining the treatment solutions. At harvest, plants are weighed, cutup, and 1 or 2 g samples are frozen immediately in liquid nitrogen andkept in a -20° C. freezer until analysis.

In the experiment with sulfonylurea-resistant Stellaria media, plantsare sprayed with one of the following treatments: 250 g/ha(dimethylphosphinyl)glycolic acid; 250 g/ha imazapyr; 10 g/hachlorsulfuron; 250 g/ha (dimethylphosphinyl)glycolic acid plus 250 g/haimazapyr; and 250 g/ha (dimethylphosphinyl)glycolic acid plus 20 g/hachlorsulfuron; with a moving belt sprayer in a spray volume of 400 L/ha.Plants are kept in a greenhouse until the tissue is extracted to measureAL and AHB levels.

Extraction and Assay Procedures

Plant tissue is pulverized in liquid nitrogen with sand and then groundin ice cold 100 mM phosphate buffer, pH 7.5 (2 mL/g). The mixture isstrained through several layers of cheesecloth into a 50 mL centrifugetube, centrifuged at 24000 g for 15 minutes and the supernatantcollected for analysis.

A portion of the supernatant is assayed directly for acetoin and anotherportion is acidified to 1% H₂ SO₄ (v/v) and heated for 15 minutes at 60°C. to decarboxylate the AL and AHB. The resulting products are assayedusing the method of Westerfeld and the combined product concentration ismeasured. There is no Westerfeld-positive product in untreated tissuebased on differences between extracts before and after acidification.Because of this, the OD₅₂₀ of extracts from untreated tissue is used tocorrect for background color.

Results

Concentrations of (dimethylphosphinyl)glycolic acid above 1 μM cause anaccumulation of AL and AHB in excised corn shoots within 8 hours afterapplication (Table I). Maximum levels appear to accumulate in plantstreated with 10 μM to 100 μM solutions of (dimethylphosphinyl)glycolicacid. A time course of this accumulation in excised corn shoots showsthat a maximum level is reached by 4 hours after application (Table II).This accumulation can be completely prevented by simultaneously treatingthe shoots with a 10 μM solution of imazaquin.(Dimethylphosphinyl)glycolic acid causes an accumulation of AL and AHBin excised sunflower shoots which can be prevented by imazaquintreatment (Table III).

Other AHAS inhibitors such as sulfometuron-methyl in the presence of theamino acids valine and leucine (and isoleucine) also prevent theaccumulation of AL and AHB caused by (dimethylphosphinyl)glycolic acid(Table IV).

The interaction between (dimethylphosphinyl)glycolic acid and AHASinhibitors can also be used to determine if a plant is resistant to theAHAS inhibitors. (Dimethylphosphinyl)glycolic acid causes anaccumulation of AL and AHB in chlorsulfuron-resistant S. media. The(dimethylphosphinyl)glycolic acid-induced accumulation of AL and AHB isprevented by imazapyr (Table V).

These results also indicate that this relatively easy method can be usedto differentiate between AHAS inhibitor resistant and susceptiblebiotypes by treating plants with a combination of(dimethylphosphinyl)glycolic acid and different AHAS inhibitors. If thebiotype is resistant to the AHAS inhibitor, then it will continue toaccumulate AL and AHB in the presence of the inhibitor. These data alsoshow that the interaction between AHAS and KARI inhibitors can be usedto study the in vivo inhibition of AHAS by monitoring the buildup of ALand AHB in the plant tissue.

In Tables I-V, the AL/AHB levels are given in terms of the opticaldensity reading at 520 nanometers (OD₅₂₀) per gram of fresh weight.

                  TABLE I    ______________________________________    Effect of different concentrations of (dimethyl-    phosphinyl)glycolic acid on AL and AHB    accumulation in excised corn shoots    eight hours after application    Concentration of    (Dimethylphosphinyl)-    glycolic acid    (μM)         AL/AHB Levels    ______________________________________    0.1             0.0    0.25            0.0    1               1.8    2.5             9.7    10              23.4    25              26.2    100             31.7    ______________________________________

                  TABLE II    ______________________________________    Time course of AL and AHB accumulation in    excised corn shoots                       AL/AHB Levels                       Time after treatment               Conc.   (hours)    Treatment    (μM)   2      4    6     8    ______________________________________    (Dimethylphosphinyl)-                 100       6.9    9.7  14.2  14.7    glycolic acid    Imazaquin     10       0      0    0     0    Imazaquin +  10 + 100  0      0    0     0    (Dimethylphosphinyl)-    glycolic acid    ______________________________________

                  TABLE III    ______________________________________    Effect of (dimethylphosphinyl)glycolic acid alone    and in combination with imazaquin on AL and AHB    accumulation in excised sunflower shoots                    Conc.    Treatment       (μM)  AL/AHB Levels    ______________________________________    Dimethylphosphinyl)-                    100      0.3    glycolic acid    (Dimethylphosphinyl)-                    100 + 10 0.05    glycolic acid +    Imazaquin    ______________________________________

                  TABLE IV    ______________________________________    Effect of (dimethylphosphinyl)glycolic acid and    various AHAS inhibitors on accumulation of AL and    AHB in excised corn shoots                      Conc.      AL/AHB    Treatment         (μM)    Levels    ______________________________________    (Dimethylphosphinyl)glycolic                      100        18    acid    (Dimethylphosphinyl)glycolic                      100 + 10 + 100                                 1    acid + Valine + Leucine    (Dimethylphosphinyl)glycolic                      100 + 1    0    acid + Sulfometuron-methyl    ______________________________________

                  TABLE V    ______________________________________    Effect of (dimethylphosphinyl)glycolic acid alone    and in combination with various AHAS inhibitors    on AL and AHB accumulation in chlorsulfuron    resistant S. media                            AL/AHB Levels                            Hours after                   Rate     treatment    Treatment        (g/ha)     24      48    ______________________________________    (Dimethylphosphinyl)glycolic                     250        25      53    acid    Chlorsulfuron     10        0       0    Imazapyr         250        0       0    (Dimethylphosphinyl)glycolic                     250 + 10   5       25    acid + Chlorsulfuron    (Dimethylphosphinyl)glycolic                     250 + 250  0       0    acid + Imazapyr    ______________________________________

EXAMPLE 2 Evaluation of In Vivo Inhibition of Acetohydroxyacid Synthasein Excised Lima Bean Shoots

Young lima bean shoots with primary leaves that are approximately 30%expanded are excised and the cut stem is placed in a 5 μM imazaquinsolution, a 100 μM (dimethylphosphinyl)glycolic acid solution, a 100 μMsodium N-hydroxy-N-isopropyloxamate solution, or a 5 μM imazaquin and100 μM sodium N-hydroxy-N-isopropyloxamate solution. The excised shootsplus solutions are placed in a lighted growth chamber (28° C.) for 7hours. After this incubation the primary leaves are harvested, frozenovernight at -20° C. and then extracted in boiling water and the levelof AL and AHB measured in the water using the Westerfeld reaction.

As can be seen from the data in Table VI, sodiumN-hydroxy-N-isopropyloxamate causes an accumulation of AL and AHB inexcised lima bean shoots which can be prevented by imazaquin treatment.

In Table VI, the AL/AHB levels are reported in terms of the opticaldensity reading at 520 nanometers (OD₅₂₀) per gram of fresh weight.

                  TABLE VI    ______________________________________    Effect of sodium N-hydroxy-N-isopropyloxamate alone    and in combination with imazaquin on AL and AHB    accumulation in excised lima bean shoots                        Conc.   AL/AHB    Treatment           (μM) Levels    ______________________________________    Imazaquin            5      0.0    (Dimethylphosphinyl)glycolic                        100     16.6    acid    Sodium N-hydroxy-N-isopropyl-                        100     13.2    oxamate    Sodium N-hydroxy-N-isopropyl-                        100 + 5 2.1    oxamate + Imazaquin    ______________________________________

EXAMPLE 3 Evaluation of In Vivo Inhibition of Acetohydroxyacid Synthasein Susceptible and Heterozygous-resistant Tobacco

Leaf discs (7 mm diameter) are cut from young expanding leaves ofimazaquin susceptible and heterozygous-resistant tobacco (Nicotianatabacum). Fifteen discs from each species are floated on 10 mL of a 5 μMimazaquin solution, a 100 μM (dimethylphosphinyl)glycolic acid solution,a 100 μM sodium N-hydroxy-N-isopropyloxamate solution, a 5 μM imazaquinand 100 μM (dimethylphosphinyl)glycolic acid solution, or a 5 μMimazaquin and 100 μM sodium N-hydroxy-N-isopropyloxamate solutioncontained in a petri plate. The discs plus solutions are incubated in alighted growth chamber for . .16.!. .Iadd.24 .Iaddend.hours. Then thediscs are harvested, frozen on dry ice and extracted in boiling water.The AL and AHB level in the water is determined using the Westerfeldreaction.

As can be seen from the data in Table VII, (dimethylphosphinyl)glycolicacid and sodium N-hydroxy-N-isopropyloxamate cause an accumulation of ALand AHB in tobacco leaf discs. This accumulation can be prevented insusceptible tobacco by imazaquin treatment. And AL and AHB accumulationin heterozygous-resistant tobacco can be prevented up to 50% byimazaquin treatment.

In Table VII, A designates imazaquin susceptible tobacco and Bdesignates imazaquin heterozygous-resistant tobacco.

In Table VII, the AL/AHB levels are reported in terms of the opticaldensity reading at 520 nanometers (OD₅₂₀) per . .gram.!. .Iadd.0.1 gram.Iaddend.of fresh weight.

                  TABLE VII    ______________________________________                           AL/AHB Levels                   Conc.   Tobacco Species    Treatment        (μM)   A       B    ______________________________________    Imazaquin         5        0.0     0.0    (Dimethylphosphinyl)glycolic                     100       2.7     2.2    acid    Sodium N-hydroxy-N-isopro-                     100       2.1     1.8    pyloxamate    (Dimethylphosphinyl)-                     100 + 5   0.0     1.2    glycolic acid + Imazaquin    Sodium N-hydroxy-N-isopro-                     100 + 5   0.2     1.0    pyloxamate + Imazaquin    ______________________________________

EXAMPLE 4 Evaluation of In Vivo Inhibition of Acetohydroxyacid Synthasein Various Weed Species

Samples of imazaquin susceptible crabgrass, morning glory andJohnsongrass and imazaquin resistant cocklebur are treated with a 0.2%imazaquin solution, a 0.1% (dimethylphosphinyl)glycolic acid solution ora 0.2% imazaquin and 0.1% (dimethylphosphinyl)glycolic acid solution.Approximately one gram of the material from each of the treatments isplaced in a test tube and 5 mL of water is added. The test tubes withthe leaf material plus water are heated in a boiling water bath for 15minutes. Three to 150 μL aliquots of the water extract are placed inseparate wells of a 96-well microtiter plate for each treatment.Twenty-five μL of 5% H₂ SO₄ is added to each well and the plate isheated at 60° C. for 15 minutes. . .Seventy-five.!. .Iadd.Sixty Two.Iaddend.μL each of 0.5% creatine in water and 5% α-naphthol in 4N NaOHis added to each well and the plate is heated at 60° C. for 15 minutes.The plate is centrifuged for . .10.!. .Iadd.15 .Iaddend.minutes at 3000g and the OD₅₂₀ is read for each well on a microtiter plate reader. Theresults are summarized in Table VIII.

As can be seen from the data in Table VIII, (dimethylphosphinyl)glycolicacid causes an accumulation of AL and AHB in various weed species. Thisaccumulation can be prevented in crabgrass, morning glory andJohnsongrass by imazaquin treatment. The accumulation of AL and AHB inthe cocklebur plant was reduced by one-half by imazaquin treatment.Therefore, these results indicate that the cocklebur plant is resistantto imazaquin.

In Table VIII, the acetolactate and acetohydroxybutyrate levels arereported in terms of the optical density reading at 520 nanometers(OD₅₂₀) per . .gram.!. .Iadd.0.15 gram .Iaddend.of fresh weight.

                  TABLE VIII    ______________________________________             Acetolactate/Acetohydroxy-             butyrate Levels             Weed Species               Crab-   Morning   Johnson-    Treatment  grass   glory     grass   Cocklebur    ______________________________________    Imazaquin  0       0         0       0    (Dimethyl- 1.7     1.5       0.5     1.0    phosphinyl)glycolic    acid    (Dimethyl- 0       0         0       0.5    phosphinyl)glycolic    acid + Imazaquin    ______________________________________

EXAMPLE 5 Evaluation of In Vivo Inhibition of Acetohydroxyacid Synthasein Imazaquin Susceptible and Resistant Cocklebur (Xanthium Strumarium)

Imazaquin susceptible and resistant cocklebur plants are treated in thefield with a 0.1% imazaquin solution, a 0.1%(dimethylphosphinyl)glycolic acid solution or a 0.1% imazaquin and 0.1%(dimethylphosphinyl)glycolic acid solution. One or two days aftertreatment, the young rapidly expanding leaves are collected.Approximately one gram of the leaf material from each of the treatmentsis placed in a test tube and 5 mL of water is added. The test tubes withthe leaf material plus water are heated in a boiling water bath for 15minutes. Three to 150 μL aliquots of the water extract are placed inseparate wells of a 96-well microtiter plate for each treatment.Twenty-five μL of 5% H₂ SO₄ is added to each well and the plate isheated at 60° C. for 15 minutes. . .Seventy-five.!. .Iadd.Fifty.Iaddend.μL each of 0.5% creatine in water and .Iadd.75 μL .Iaddend.5%α-naphthol in 4N NaOH is added to each well and the plate is heated at60° C. for 15 minutes. The plate is centrifuged for 10 minutes at 3000 gand the . .OD₅₂₀ .!. .Iadd.OD₅₃₅ .Iaddend.is read for each well on amicrotiter plate reader. The results are summarized in Table IX.

As can be seen from the data in Table IX, (dimethylphosphinyl)glycolicacid causes an accumulation of AL and AHB in both cocklebur plants. Thisaccumulation can be prevented in the imazaquin susceptible cockleburplant. However, the accumulation of AL and AHB in the other cockleburplant could not be completely prevented by imazaquin treatment.Therefore, these results indicate that this cocklebur plant is resistantto imazaquin.

                  TABLE I    ______________________________________                 AL/AHB Levels                 Cocklebur Plant    Treatment      Imazaquin-Susceptible    Resistant    ______________________________________    (Dimethylphosphinyl)-                   3.42          3.44    glycolic acid    (Dimethylphosphinyl)-                   0.04          1.39    glycolic acid +    Imazaquin    ______________________________________

I claim:
 1. An in vivo method for determining whether a compoundinhibits acetohydroxyacid synthase which comprises:(a) treating a firstacetohydroxyacid synthase inhibitor-susceptible plant or plant part withan effective amount of the compound and an effective amount of aketol-acid reductoisomerase inhibitor; (b) treating a secondacetohydroxyacid synthase inhibitor-susceptible plant or plant part fromthe same population of the same species with the effective amount of theketol-acid reductiosomerase inhibitor alone; and (c) measuring theamounts of acetolactate and acetohydroxybutyrate present in the treatedplants or plant parts to determine if the amount of acetolactate andacetohydroxybutyrate present in (a) is less than the amount ofacetolactate and acetohydroxybutyrate present in (b).
 2. The methodaccording to claim 1 wherein the ketol-acid reductoisomerase inhibitoris selected from the group consisting of (dimethylphosphinyl)glycolicacid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, sodiumN-hydroxy-N-alkyloxamate and sodium N-hydroxy-N-aralkyloxamate.
 3. Themethod according to claim 2 wherein the alkyl component of theN-hydroxy-N-alkyloxamate is selected from C₁ -C₆ alkyl and C₃ -C₇cycloalkyl.
 4. The method according to claim 3 wherein the alkylcomponent is isopropyl.
 5. The method according to claim 2 wherein thearalkyl component is benzyl.
 6. The method according to claim 1 whereinthe acetohydroxyacid synthase inhibitor susceptible plant is selectedfrom the group consisting of a monocotyledonous plant and adicotyledonous plant.
 7. The method according to claim 1 wherein theplant parts are young rapidly growing tissue.
 8. The method according toclaim 1 wherein the amount of acetolactate and acetohydroxybutyratepresent in the treated plant or plant part is determined by:(a)extracting the acetolactate and acetohydroxybutyrate present in thetreated plant or plant part into water; (b) treating the water extractwith a sulfuric acid solution; (c) treating the acidified water extractsolution from (b) with a 0.5% creatine solution and a 5% α-naphthol insodium hydroxide solution; and (d) measuring and comparing the color ofthe product of (c) to a known standard.
 9. The method according to claim1 wherein the plant or plant part is treated with a 1 μM to 1,000 μMsolution of the compound and a 1 μM to 1,000 μM solution of theketol-acid reductoisomerase inhibitor.
 10. An in vivo method fordetermining whether a population of a plant species is resistant to anacetohydroxyacid synthase inhibitor which comprises:(a) treating a firstplant or a part of the plant with an effective amount of theacetohydroxyacid synthase inhibitor and an effective amount of aketol-acid reductoisomerase inhibitor; (b) treating a second plant orplant part from the same population of the same species with theeffective amount of the ketol-acid reductoisomerase inhibitor alone; (c)measuring the amounts of acetolactate and acetohydroxybutyrate presentin the treated plants or plant parts to determine if the amount ofacetolactate and acetohydroxybutyrate present in (a) is at least about15% of the amount of acetolactate and acetohydroxybutyrate present in(b).
 11. The method according to claim 10 wherein the acetohydroxyacidsynthase inhibitor is selected from the group consisting of animidazolinone, a sulfonylurea, a sulfonamide and a pyrimidyloxybenzoate.12. The method according to claim 11 wherein the imidazolinone isselected from the group consistingof5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinicacid;2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylicacid; isopropylammonium2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate; methyl2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate; and2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid.13. The method according to claim 10 wherein the ketol-acidreductoisomerase inhibitor is selected from the group consisting of(dimethylphosphinyl)glycolic acid,2-(dimethylphosphinoyl)-2-hydroxyacetic acid, sodiumN-hydroxy-N-alkyloxamate and sodium N-hydroxy-N-aralkyloxamate.
 14. Themethod according to claim 13 wherein the alkyl component of theN-hydroxy-N-alkyloxamate is selected from C₁ -C₆ alkyl and C₃ -C₇cycloalkyl.
 15. The method according to claim 14 wherein the alkylcomponent is isopropyl.
 16. The method according to claim 13 wherein thearalkyl component is benzyl.
 17. The method according to claim 10wherein the plant parts are young rapidly growing tissue.
 18. The methodaccording to claim 10 wherein the amount of acetolactate andacetohydroxybutyrate present in the treated plant or plant part isdetermined by(a) extracting the acetolactate and acetohydroxybutyratepresent in the plant or plant part into water; (b) treating the waterextract with a sulfuric acid solution; (c) treating the acidified waterextract solution from (b) with a 0.5% creatine solution and a 5%α-naphthol in sodium hydroxide solution; and (d) measuring and comparingthe color of the product of (c) to a known standard.
 19. The methodaccording to claim 10 wherein the plant or plant part is treated with a1 μM to 1,000 μM solution of the acetohydroxyacid synthase inhibitor anda 1 μM to 1,000 μM solution of the ketol-acid reductoisomeraseinhibitor. .Iadd.
 20. A method for determining whether a material to betested is capable of inhibiting acetolactate synthesis in a given planttissue sample containing living cells which comprises the steps of:(a)combining in an aqueous medium the plant tissue sample, an effectiveamount of the material, and an effective amount of an inhibitor of ketoacid reductoisomerase, so that acetolactate will accumulate in themixture unless the material inhibits acetolactate synthesis; and (b)detecting accumulation of acetolactate..Iaddend..Iadd.21. The methodaccording to claim 20 wherein the keto acid reductoisomerase inhibitoris selected from the group consisting of (dimethylphosphinyl)glycolicacid, 2-(dimethylphosphinoyl)-2-hydroxyacetic acid, sodiumN-hydroxy-N-alkyloxamate and sodiumN-hydroxy-N-aralkyloxamate..Iaddend..Iadd.22. The method according toclaim 21 wherein the alkyl component of the N-hydroxy-N-alkyoxamate isselected from C₁ -C₆ alkyl and C₃ -C₇ cycloalkyl..Iaddend..Iadd.23. Themethod according to claim 22 wherein the alkyl component isisopropyl..Iaddend..Iadd.24. The method according to claim 21 whereinthe aralkyl component is benzyl..Iaddend..Iadd.25. The method accordingto claim 20 wherein the given plant tissue sample is selected from thegroup consisting of a monocotyledonous plant and a dicotyledonousplant..Iaddend..Iadd.26. The method according to claim 20 wherein thegiven plant tissue sample is young rapidly growingtissue..Iaddend..Iadd.27. The method according to claim 20 wherein step(b), detecting accumulation of acetolactate, comprises the steps of:(c)extracting the acetolactate present in the given plant tissue sampleinto water; (d) treating the water extract with a sulfuric acidsolution; (e) treating the acidified water extract solution from (d)with a 0.5% creatine solution and a 5% α-naphthol in sodium hydroxidesolution; and (f) measuring and comparing the color of the product of(e) to a known standard..Iaddend..Iadd.28. The method according to claim20 wherein the given plant tissue sample is treated with a 1 μM to 1,000μM solution of the material and a 1 μM to 1,000 μM solution of the ketoacid reductoisomerase inhibitor..Iaddend..Iadd.29. A method of claim 20wherein step (b), detecting accumulation of acetolactate, comprises thesteps of: (c) allowing time for acetolactate to accumulate; (d)rupturing the cells; (e) acidifying said mixture to convert anyaccumulated acetolactate to acetoin, and (f) colorimetrically detectingthe presence of acetoin in the mixture..Iaddend..Iadd.30. The method ofclaim 29 wherein step (f) comprises the step of adding an effectiveamount of a compound containing the guanidino group, 1-naphthol, andbase to the mixture..Iaddend..Iadd.31. The method of claim 30 whereinthe compound containing the guanidino group iscreatine..Iaddend..Iadd.32. A method for determining whether a givenplant is resistant to a herbicide known to have inhibition ofacetolactate synthase as its mode of action, which comprises:(a)combining in an aqueous medium a fresh sample of tissue from said plant,an effective amount of said herbicide, and an effective amount of a ketoacid reductoisomerase inhibitor; and (b) detecting the accumulation ofacetolactate..Iaddend..Iadd.33. The method according to claim 32 whereinthe herbicide is an acetohydroxyacid synthase inhibitor selected fromthe group consisting of an imidazolinone, a sulfonyl-urea, a sulfonamideand a pyrimidyloxybenzoate..Iaddend..Iadd.34. The method according toclaim 33 wherein the imidazolinone is selected from the group consistingof 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinicacid;2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylicacid; isopropylammonium2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate; methyl2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinate; and2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinicacid..Iaddend..Iadd.35. The method according to claim 32 wherein theketo acid reductoisomerase inhibitor is selected from the groupconsisting of (dimethylphosphinyl)glycolic acid,2-(dimethylphosphinoyl)-2-hydroxyacetic acid, sodiumN-hydroxy-N-alkyloxamate and sodiumN-hydroxy-N-aralkyloxamate..Iaddend..Iadd.36. The method according toclaim 35 wherein the alkyl component of the N-hydroxy-N-alkyloxamate isselected from C₁ -C₆ alkyl and C₃ -C₇ cycloalkyl..Iaddend..Iadd.37. Themethod according to claim 35 wherein the alkyl component isisopropyl..Iaddend..Iadd.38. The method according to claim 35 whereinthe aralkyl component is benzyl..Iaddend..Iadd.39. The method accordingto claim 32 wherein the given plant tissue sample is young rapidlygrowing tissue..Iaddend..Iadd.40. The method according to claim 32wherein step (b), detecting the accumulation of acetolactate, comprisesthe steps of:(c) extracting the acetolactate present in the given planttissue sample into water; (d) treating the water extract with a sulfuricacid solution; (e) treating the acidified water extract solution from(d) with a 0.5% creatine solution and a 5% α-naphthol in sodiumhydroxide solution; and (f) measuring and comparing the color of theproduct of (e) to a known standard..Iaddend..Iadd.41. The methodaccording to claim 32 wherein the given plant tissue sample is treatedwith a 1 μM to 1,000 μM solution of the acetohydroxyacid synthaseinhibitor and 1 μM to 1,000 μM solution of the keto acidreductoisomerase inhibitor..Iaddend..Iadd.42. The method of claim 32wherein said herbicide is known to have inhibition of acetolactatesynthase as its mode of action..Iaddend..Iadd.43. The method of claim 32wherein the herbicide is selected from the group consisting offlumetsulam, imazaquin, chlorsulfuron, sulfometuron-methyl, imazapyr,and imazethapyr..Iaddend..Iadd.44. The method of claim 32 wherein step(b), detecting the accumulation of acetolactate, comprises the steps of:(c) rupturing the cells of said plant tissue sample; (d) acidifying saidmixture, formed by the combining step (a), to convert any accumulatedacetolactate to acetoin; and (e) adding effective amounts of a compoundcontaining the guanidino group, 1-naphthol, and base to the mixture, sothat the color of the resulting mixture indicates whether acetolactatesynthesis was inhibited..Iaddend..Iadd.45. The method of claim 44wherein the compound containing the guanidino group iscreatine..Iaddend.